Abstract:

It has been discovered that a column configuration which allows for
suspension of the beads, such as a mobile bead, during washing results in
significantly lower levels of background and hence more sensitive levels
of quantitation. The present invention provides columns, kits, and
methods for more sensitive detection of a toxin or other analyte.

Claims:

1. A method of analyzing a single liquid sample for detecting the presence
of an analyte in a sample or for analyzing the level of an analyte in a
sample, wherein the method comprises:a. providing an immunoaffinity
column comprising a mobile resin configuration, wherein the
immunoaffinity column comprises:i. a lower end including an outflow
opening;ii. a lower porous support;iii. a layer of resin on the lower
porous support, the resin having specific affinity for the analyte;iv. an
upper porous support; andv. an upper end including an inflow
opening;wherein the resin between the lower porous support and the upper
porous support is structured and arranged to permit removal of the upper
frit from the column without substantial removal of resin;b. loading the
column with a predetermined amount of a liquid sample suspected of
containing the analyte;c. binding the analyte to the antibody on the
column;d. loading the column with a wash solutione. suspending the resin
in the wash solution;f. removing the wash solution from the column;g.
eluting the analyte in eluant; andh. analyzing the eluant for the
presence of the analyte.

2. The method of claim 1, wherein the volume of the resin in the column is
less than the total packed volume of the column between the lower porous
support and the upper porous support.

3. The method of claim 1, wherein the volume of the resin in the column is
no greater than 50% of the total packed volume of the column between the
lower porous support and the upper porous support.

4. The method of claim 1, wherein the volume of the resin in the column is
no greater than about 40% of the total packed volume of the column
between the lower porous support and the upper porous support.

5. The method of claim 1, wherein the volume of the resin in the column is
no greater than about 30% of the total packed volume of the column
between the lower porous support and the upper porous support.

6. The method of claim 1, wherein the volume of the resin in the column is
no greater than about 25% of the total packed volume of the column
between the lower porous support and the upper porous support.

7. The method of claim 1, wherein the volume of the resin in the column is
no greater than about 20% of the total packed volume of the column
between the lower porous support and the upper porous support.

8. The method of claim 1, wherein the column is structured and arranged to
recover at least 60% of the analyte in a 40 ml sample being analyzed, the
analyte being selected from the group consisting of ochratoxin,
zearalenone, aflatoxin, fumonisin, T-2, HT-2, and deoxynivalenol.

9. The method of claim 8, wherein the column is structured and arranged to
recover at least about 70% of the analyte in a 40 ml sample being
analyzed.

10. The method of claim 8, wherein the column is structured and arranged
to recover at least about 80% of the analyte in a 40 ml sample being
analyzed.

11. The method of claim 1, wherein the sample comprises milk or a milk
product and the analyte is an aflatoxin.

13. The method of claim 11, wherein the aflatoxin is aflatoxin M.sub.1.

14. The method of claim 1, wherein the column has a detection limit at or
below about 100 ppt.

15. The method of claim 14, wherein the column has a detection limit at or
below about 50 ppt.

16. The method of claim 14, wherein the column has a detection limit at or
below about 25 ppt.

17. The method of claim 14, wherein the column has a detection limit at or
below about 15 ppt.

18. The method of claim 14, wherein the column has a detection limit at or
below about 12.5 ppt.

19. The method of claim 1, wherein the column is structured and arranged
to have a flow rate of sample fluid through the column of at least 1-2
drops per second.

20. The method of claim 1, wherein the resin comprises about 5 mg of
antibody per ml of resin.

21. The method of claim 1, wherein the column further comprises at least
one of the following:a. a removable cap covering the lower end to prevent
transfer of substances through the outflow opening; orb. a removable cap
covering the upper end to prevent transfer ofsubstances through the
inflow opening; andand wherein the resin is suspended by at least one
inversion of the column.

22. The method of claim 1, wherein the analyte is extracted from a food
using a water-based or water compatible solvent.

23. The method of claim 22, wherein the food comprises a milk product.

24. The method of claim 1, wherein the analyte is extracted from milk or a
milk product.

25. The method of claim 24, wherein the analyte is extracted from
de-fatted milk.

26. The method of claim 1, wherein the resin is suspended by inversion of
the column, by vortexing the column, or by shaking the column.

27. The method of claim 1, wherein the resin is suspended by inverting the
column until the resin is released from the lower porous support.

28. The method of claim 1, wherein the upper porous support is removed
from the column following the step of binding the analyte to the antibody
on the column.

29. An immunoaffinity column comprising a mobile resin configuration for
detecting the presence of an analyte in a sample or for analyzing the
level of an analyte in a sample, wherein the immunoaffinity column
comprises:a. a lower end including an outflow opening;b. a lower porous
support;c. a layer of resin on the lower porous support, the resin having
specific immunoaffinity for the analyte;d. an upper porous support; ande.
an upper end including an inflow opening;wherein the resin between the
lower porous support and the upper porous support is structured and
arranged to permit removal of the upper frit from the column without
substantial removal of resin.

30-49. (canceled)

50. A kit for detecting the presence of an analyte in a sample or for
analyzing the level of a analyte in a sample, wherein the kit
comprises:a. the immunoaffinity column of claim 29 having a mobile resin
configuration;b. a removable cap covering the lower end to prevent
transfer of substances through the outflow opening;c. a removable cap
covering the upper end to prevent transfer of substances through the
inflow opening; andd. instructions for use.

[0002]The invention relates to affinity columns such as those used for
immunological screening for environmentally occurring toxins, for
example, those toxins found in food products, and is particularly useful
for detecting Aflatoxins with high sensitivity in dairy products. It has
been discovered that a column configuration which allows for suspension
of the beads, such as a mobile bead, during washing results in
significantly lower levels of background and hence more sensitive levels
of quantitation. The present invention provides columns, kits, and
methods for more sensitive detection of a toxin or other analyte.

BACKGROUND OF THE INVENTION

[0003]The incidence and effect of exposure to toxic substances by humans
and other animals via food, water, and air is of critical importance to
our survival. The detection of toxins such as aflatoxin, ochratoxin,
zearalenone, deoxynivalenol and fumonisin has become especially
important. In particular, screening procedures for assessing the exposure
of humans to such toxins may require the ability to quantify both the
toxin and its metabolites. There have been numerous reported incidences
of naturally occurring mycotoxins such as, aflatoxin B1, B2,
G1, G2 and M1 (Afla), deoxynivalenol (DON), fumonisin
B1, B2 and B3, ochratoxin A (OTA), and zearalenone (Zear)
in various substrates. Malt beverages and wines can contain different
multi-toxin combinations from fungi-infected grains and fruits used in
the production. Milk, tea, and coffee can also contain some of these
toxins.

[0004]Aflatoxins are a typical example of the compounds for which
screening is desired. Aflatoxins are secondary fungal metabolites,
mycotoxins, which are produced by Aspergillus flavus and Aspergillus
parasiticus and are structurally a group of substituted coumarins
containing a fused dihydrofurofuran moiety. Aflatoxins occur naturally in
peanuts, peanut meal, cottonseed meal, corn, dried chili peppers, and the
like. However, the growth of the mold itself does not predict the
presence or levels of the toxin because the yield of aflatoxin depends on
growth conditions as well as the genetic requirements of the species. A
variety of aflatoxins, that is types B1, B2, G1, G2,
M1 and M2, have been isolated and characterized. Aflatoxin
B1 ("AFB1") is the most biologically potent of these aflatoxins
and has been shown to be toxic, mutagenic and carcinogenic in many animal
species. This mycotoxin is a frequent contaminant of the human food
supply in many areas of the world and is statistically associated with
increased incidence of human liver cancer in Asia and Africa, in
particular (Busby et al., in Food-Born Infections and Intoxications
(Riemann and Bryan, Editors) Second Edition, Academic Press, Inc., 1979,
pp. 519-610; Wogan, G. N. Methods Cancer Res. 7:309-344 (1973)).

[0006]U.S. Pat. No. 4,818,687 (granted Apr. 4, 1989) and U.S. Pat. No.
4,859,611 (granted Aug. 22, 1989) describe a general non-invasive
screening procedure for assessing the exposure of humans and animals to
environmentally occurring carcinogens. Therein, an affinity matrix and a
method for the detection of low molecular weight compositions, such as
aflatoxins, are provided utilizing specific monoclonal IgM antibody.

[0007]AFB1 found in feed may also be ingested by dairy cows and
subsequently metabolized to aflatoxin M1 ("AFM1"). AFM1 is
approximately 75% as toxic to ducklings as AFB1 (Purchase, I. F. H.,
Food Cosmet. Toxicol., 5:339-342 (1969)). Consequently, many countries
regulate the maximum permissible of AFM1 found in milk and milk
products. The action level for AFM1 in milk established by the US
Food and Drug Administration is 0.5 ng/ml (500 parts per trillion, 500
ppt). The European Union regulates AFM1 in milk more stringently at
50 ppt for adults and 25 ppt for infant formula. Raw milk is a colloidal
suspension

[0009]Methods relying on extraction of AFM1 by immunoaffinity columns
have also been published. This approach circumvents some of the slow and
laborious preparation of the methods given above. However, quantitation
of the AFM1 by HPLC after immunoaffinity cleanup (Dragacci, S, and
Grosso JOAOC 84:437-443 (2001)) still requires expensive equipment and a
certain degree of technical expertise. Quantitation by fluorometer after
immunoaffinity cleanup (Hansen, T. J. Food Protection 53:75-77 (1990))
requires less expensive equipment, is faster and simpler, but has a
reported detection limit of 50 ppt which is not adequate when
quantitation is required at lower concentrations than 50 ppt.

[0010]The limit of quantitation using a fluorometer has been constrained
by the level of fluorescent background materials not removed by the
immunoaffinity column purification. Typically in such a column antibody
bound to a resin or other solid medium is held between two porous frits
and non-specifically bound fluorescent background material is removed by
washing with an appropriate buffer. Pockets or bubbles of air or other
gases may become trapped within the resin and may reduce efficient
washing of the resin. It is believed that these entrapped pockets or
bubbles may reduce the sensitivity of the column.

[0011]There is a need for a more sensitive method of measuring
contaminants in a liquid or colloidal sample, particularly one that is
relatively rapid, simple, and inexpensive, such as quantitation of
aflatoxin in milk using a fluorometer after immunoaffinity column
purification.

SUMMARY OF THE INVENTION

[0012]In one aspect, the present invention provides a method of analyzing
a single liquid sample for detecting the presence of an analyte in a
sample or for analyzing the level of an analyte in a sample, wherein the
method comprises: [0013]a. providing an immunoaffinity column
comprising a mobile resin configuration, wherein the immunoaffinity
column comprises: [0014]i. a lower end including an outflow opening;
[0015]ii. a lower porous support; [0016]iii. a layer of resin on the
lower porous support, the resin having specific affinity for the analyte;
[0017]iv. an upper porous support; and [0018]v. an upper end including an
inflow opening; [0019]wherein the resin between the lower porous
support and the upper porous support is structured and arranged to permit
removal of the upper frit from the column without substantial removal of
resin; [0020]b. loading the column with a predetermined amount of a
liquid sample suspected of containing the analyte; [0021]c. binding the
analyte to the antibody on the column; [0022]d. loading the column with a
wash solution [0023]e. suspending the resin in the wash solution;
[0024]f. removing the wash solution from the column; [0025]g. eluting the
analyte in eluant; and [0026]h. analyzing the eluant for the presence of
the analyte.

[0027]In one embodiment, the volume of the resin in the column is less
than the total packed volume of the column between the lower porous
support and the upper porous support.

[0028]In one embodiment, the column is structured and arranged to recover
at least 60% of the analyte in a 40 ml sample being analyzed, the analyte
being selected from the group consisting of ochratoxin, zearalenone,
aflatoxin, fiimonisin, T-2, HT-2, and deoxynivalenol.

[0029]In various embodiments, the column has a detection limit at or below
about 100 ppt, 50 ppt, 25 ppt, 15 ppt, or 12.5 ppt.

[0030]In another aspect, the present invention provides an immunoaffinity
column comprising a mobile resin configuration for detecting the presence
of an analyte in a sample or for analyzing the level of an analyte in a
sample, wherein the immunoaffinity column comprises: [0031]a. a lower
end including an outflow opening; [0032]b. a lower porous support;
[0033]c. a layer of resin on the lower porous support, the resin having
specific immunoaffinity for the analyte; [0034]d. an upper porous
support; and [0035]e. an upper end including an inflow opening;wherein
the resin between the lower porous support and the upper porous support
is structured and arranged to permit removal of the upper frit from the
column without substantial removal of resin.

[0036]In yet another aspect, the present invention provides a kit for
detecting the presence of an analyte in a sample or for analyzing the
level of an analyte in a sample, wherein the kit comprises: [0037]a.
the immunoaffinity column described above and having a mobile resin
configuration; [0038]b. a removable cap covering the lower end to prevent
transfer of substances through the outflow opening; [0039]c. a removable
cap covering the upper end to prevent transfer of substances through the
inflow opening; and [0040]d. instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041]FIG. 1 is a schematic cross-section depicting an embodiment of the
mobile bead column of the present invention.

[0042]FIG. 2 is a graph showing the linear regression analysis equation
for the amount of aflatoxin M1 measured by fluorometer (y axis)
versus the amount spiked (x axis) from analysis of spiked samples using a
mobile bead column of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0043]It has been discovered that a column configuration which allows for
suspension of the beads, such as a mobile bead, during washing results in
significantly lower levels of background and hence more sensitive levels
of quantitation. The present invention provides columns, kits, and
methods for more sensitive detection of a toxin or other analyte.

[0044]It has been discovered that a column configuration which allows for
suspension of the beads, such as a mobile bead column (e.g., a mobile
bead configuration column) or other resin during washing results in
significantly lower levels of background fluorescence and hence more
sensitive levels of quantitation.

[0045]The present invention provides an immunoaffinity column capable of
analyzing a single sample containing aflatoxin. In one embodiment, the
column in accord with the present invention comprises a quantity of resin
comprising an antibody having specificity for a toxin (e.g., aflatoxin),
the resin being fixed between two semi-porous frits such that the resin
beads may be suspended and such that the upper frit may be removed during
washing of the resin to remove fluorescent background compounds.

[0046]In a preferred embodiment, the column of the present invention is
capable of analyzing a sample to detect aflatoxins M1, G1,
G2, B1, and B2.

[0047]The invention also provides a method for analyzing a single sample
for a toxin e.g., aflatoxin), the method comprising providing a column as
described herein, applying liquid sample suspected of containing one or
more of the specified toxins to bind to resins in the column, washing the
column, eluting the resins and analyzing the eluant for the presence of
the specified toxins.

[0048]The present invention provides affinity columns, methods, and kits
used for immunological screening for environmentally occurring toxins,
for example, those found in food products, and is particularly useful for
detecting aflatoxins with high sensitivity in dairy products.

[0049]In one aspect, the present invention provides a method of analyzing
a single liquid sample for detecting the presence of an analyte in a
sample or for analyzing the level of an analyte in a sample, wherein the
method comprises: [0050]a. providing an immunoaffinity column
comprising a mobile resin configuration, wherein the immunoaffinity
column comprises: [0051]i. a lower end including an outflow opening;
[0052]ii. a lower porous support; [0053]iii. a layer of resin on the
lower porous support, the resin having specific affinity for the analyte;
[0054]iv. an upper porous support; and [0055]v. an upper end including an
inflow opening; [0056]wherein the resin between the lower porous
support and the upper porous support is structured and arranged to permit
removal of the upper frit from the column without substantial removal of
resin; [0057]b. loading the column with a predetermined amount of a
liquid sample suspected of containing the analyte; [0058]c. binding the
analyte to the antibody on the column; [0059]d. loading the column with a
wash solution [0060]e. suspending the resin in the wash solution;
[0061]f. removing the wash solution from the column; [0062]g. eluting the
analyte in eluant; and [0063]h. analyzing the eluant for the presence of
the analyte.

[0064]In one embodiment, the volume of the resin in the column is less
than the total packed volume of the column between the lower porous
support and the upper porous support. Preferably, the volume of the resin
in the column is no greater than 50%, more preferably no greater than
40%, even more preferably no greater than 30%, still more preferably no
greater than 25%, and still more preferably no greater than 20% of the
total packed volume of the column between the lower porous support and
the upper porous support.

[0065]In another embodiment, the column is structured and arranged to
recover at least about 60%, preferably at least about 70%, and more
preferably about 80% of the analyte in a 40 ml sample being analyzed, the
analyte being selected from the group consisting of ochratoxin,
zearalenone, aflatoxin, fumonisin, T-2, HT-2, and deoxynivalenol.

[0066]In yet another embodiment, the sample comprises milk or a milk
product and the analyte is an aflatoxin. Preferably, the aflatoxin is
aflatoxin G1, G2, B1, B2, or M1. More
preferably, the aflatoxin is aflatoxin M1.

[0067]In a preferred embodiment, the column has a detection limit at or
below about 100 ppt, more preferably at or below about 50 ppt, even more
preferably at or below about 25 ppt, still more preferably at or below
about 15 ppt, still more preferably at or below about 12.5 ppt.

[0068]In another embodiment, the column is structured and arranged to have
a flow rate of sample fluid through the column of at least about 1-2
drops per second.

[0069]In yet another embodiment, the resin comprises about S mg of
antibody per ml of resin.

[0070]In still another embodiment, the column further comprises at least
one of the following: [0071]a. a removable cap covering the lower end
to prevent transfer of substances through the outflow opening; or
[0072]b. a removable cap covering the upper end to prevent transfer of
substances through the inflow opening; andand wherein the resin is
suspended by at least one inversion of the column.

[0073]In one embodiment, the analyte is extracted from a food using a
water-based or water compatible solvent. Preferably, the food comprises a
milk product.

[0074]In another embodiment, the analyte is extracted from milk or a milk
product. Preferably, the analyte is extracted from de-fatted milk.

[0075]In one embodiment, the resin is suspended by inversion of the
column, by vortexing the column, or by shaking the column. Preferably,
the resin is suspended by inverting the column until the resin is
released from the lower porous support.

[0076]In yet another embodiment, the upper porous support is removed from
the column following the step of binding the analyte to the antibody on
the column.

[0077]In another aspect, the present invention provides an immunoaffinity
column comprising a mobile resin configuration for detecting the presence
of an analyte in a sample or for analyzing the level of an analyte in a
sample, wherein the immunoaffinity column comprises: [0078]a. a lower
end including an outflow opening; [0079]b. a lower porous support;
[0080]c. a layer of resin on the lower porous support, the resin having
specific immunoaffinity for the analyte; [0081]d. an upper porous
support; and [0082]e. an upper end including an inflow opening;wherein
the resin between the lower porous support and the upper porous support
is structured and arranged to permit removal of the upper frit from the
column without substantial removal of resin.

[0083]In one embodiment, the volume of the resin in the column is less
than the total packed volume of the column between the lower porous
support and the upper porous support. Preferably, the volume of the resin
in the column is no greater than 50%, more preferably no greater than
40%, even more preferably no greater than 30%, still more preferably no
greater than 25%, and still more preferably no greater than 20% of the
total packed volume of the column between the lower porous support and
the upper porous support.

[0084]In another embodiment, the column is structured and arranged to
recover at least about 60%, preferably at least about 70%, and more
preferably about 80% of the analyte in a 40 ml sample being analyzed, the
analyte being selected from the group consisting of ochratoxin,
zearalenone, aflatoxin, fumonisin, T-2, HT-2, and deoxynivalenol.

[0085]In yet another embodiment, the sample comprises milk or a milk
product and the analyte is an aflatoxin. Preferably, the aflatoxin is
aflatoxin G1, G2, B1, B2, or M1. More
preferably, the aflatoxin is aflatoxin M1.

[0086]In a preferred embodiment, the column has a detection limit at or
below about 100 ppt, more preferably at or below about 50 ppt, even more
preferably at or below about 25 ppt, still more preferably at or below
about 15 ppt, still more preferably at or below about 12.5 ppt.

[0087]In another embodiment, the column is structured and arranged to have
a flow rate of sample fluid through the column of at least 1-2 drops per
second.

[0088]In yet another embodiment, the resin comprises about 5 mg of
antibody per ml of resin.

[0089]In still another embodiment, the column further comprises at least
one of the following: [0090]a. a removable cap covering the lower end
to prevent transfer of substances through the outflow opening; or
[0091]b. a removable cap covering the upper end to prevent transfer of
substances through the inflow opening;and wherein the resin is suspended
by at least one inversion of the column.

[0092]In yet another aspect, the present invention provides a kit for
detecting the presence of an analyte in a sample or for analyzing the
level of an analyte in a sample, wherein the kit comprises: [0093]a.
the immunoaffinity column described above and having a mobile resin
configuration; [0094]b. a removable cap covering the lower end to prevent
transfer of substances through the outflow opening; [0095]c. a removable
cap covering the upper end to prevent transfer of substances through the
inflow opening; and [0096]d. instructions for use.

[0097]In one embodiment, (a) the volume of the resin is less than the
total packed volume of the column between the lower porous support and
the upper porous support; and (b) the column has a detection limit at or
below about 50 ppt.

[0098]Preferably, the volume of the resin is no greater than about 50%,
more preferably no greater than about 25%, and even more preferably no
greater than about 20% of the total packed volume of the column between
the lower porous support and the upper porous support

[0099]Preferably, the column has a detection limit at or below about 25
ppt, more preferably at or below about 15 ppt, and even more preferably
at or below about 12.5 ppt.

[0100]FIG. 1 is a schematic cross-section depicting one embodiment of the
mobile bead column of the present invention. The vertical column (10) has
an upper end (12) having an inflow opening (14) and a lower end (16)
having an outflow opening (18) such that liquids or colloids enter the
column through the inflow opening (14) and exit it through the outflow
opening (18). In a preferred embodiment, the two ends (12, 16) are fitted
with caps of appropriate dimensions and materials to prevent or inhibit
the contents of the column from exiting the column, even when the column
is inverted. These types of caps are known to those of ordinary skill in
the art.

[0101]The column has a lower frit (22) positioned at or near the base of
the column. A layer of resin (24) rests on the lower frit (22). An upper
frit (26) is positioned above the resin at a distance sufficient to
permit the particles of the resin to have a degree of mobility within the
column. The two frits (22, 26) have a porosity that enables them to
confine the resin within the discrete space while allowing inflow and
outflow of the liquid or colloidal sample.

[0102]The volume of the resin in the column is less than the total packed
volume of the column between the lower support and the upper support. The
packed volume of the column refers to the volume of the column between
the lower support and the upper support not including any spaces between
the beads when beads are packed into the portion of the column between
the lower support and the upper support. The volume of resin between the
upper and lower frits is sufficiently loose to enable removal of the
upper frit without loss of resin to a degree that would statistically
impact test results (which could yield an artificially low reading of the
toxin concentration) or inadvertent mixing of particles or precipitates
trapped on the upper frit.

[0103]For example, in one embodiment of the invention, in a 3 ml column
having a diameter of 8.93 mm, a supporting lower porous disk, or the
like, is positioned to permit flow out of the column, while another
porous disk, or the like, if desired, is positioned one inch above the
bottom of the column barrel. The positioning of the upper frit will
depend on the amount of resin required for the test and the size of the
column and can be determined without undue experimentation.

[0104]In one embodiment, a toxin (e.g., aflatoxin) is quantified in a
sample (e.g., milk). In addition to distributing the test sample, the
upper frit captures larger particles and precipitates (e.g., precipitates
from refrigeration) in the test sample. Some of these particles and
precipitates can contribute to detected background levels (e.g.,
background fluorescence) and decrease the sensitivity of the test being
performed. In this embodiment, the upper frit is removed prior to washing
to prevent the particles and/or precipitates from being solubilized by
the elution solution, followed by their elution with the eluant and
subsequent detection. For example, some particles or precipitates may be
solubilized in an 80% or higher methanol solution, thereby increasing
detected fluorescence.

[0105]The volume of a right circular cylinder is computed as
V=πr2h, where V is the volume, r is the radius of the cylinder,
and h is its height. For example, for a column having a diameter of 8.93
mm (r=0.4465 cm) with two frits positioned 1 inch (h=2.54 mm) apart, the
volume of this space is approximately 1.5908 cm3 or 1590.8 μl
(using π=3.14159). One of skill in the art would know, or would be
able to determine without undue experimentation, the relative positioning
of the frits with respect to the amount of resin loaded (for a 1-2
drop/second flow rate) in order to allow mobility of the beads or other
resin between the frits to allow for suspension and washing.

[0106]In one embodiment, the volume of resin is no greater than 50% of the
total packed volume. For example, in one embodiment of the column
described above, 100-600 μl of a resin having an antibody specific for
a toxin is layered on the disk. Preferably, the volume of resin is less
than 50% of the total packed volume, more preferably less than 40%, even
more preferably less than 30%, still more preferably less than 25%, and
even still more preferably less than 20% of the total packed volume, but
other factors could include, for example, the degree of affinity of the
resin for the toxin and the impact of the buffers and other solutions
used for washing and elution.

[0107]In accordance with the present invention, an immunoaffinity column
capable of analyzing a single sample containing aflatoxins, can be
prepared. Resins containing antibody having specificity for the toxins
are required. Antibodies are raised by well known techniques and
monoclonal antibodies are prepared having specificity for the toxin.
Resins having antibody bound thereto is prepared by techniques well known
to those skilled in the art. Any known resin material can be used. A
preferred resin material is Sepahrose 4B from Amersham Biosciences
(Piscataway, N.J.). The antibodies are then attached to the resin using
techniques well known to those skilled in the art. Preferably, for
example, about 5 mg of anti-aflatoxin antibody is bound to one ml of
resin. The resin preferably has a particle size range of about 45 to
about 165 μm.

[0108]Columns are then prepared using appropriate quantities of each
resin. For example, in one embodiment of the invention, in a 3 ml column
having a diameter of 8.93 mm, a supporting porous disk, or the like, is
positioned that permits flow out of the column. Approximately 100-600
μl of a resin having an antibody specific for a toxin is layered on
the disk. In one embodiment, 250 μl of a resin having an antibody
specific for aflatoxin is layered on the disk. Finally, another porous
disk, or the like, if desired, is positioned one inch above the bottom of
the column barrel to contain the resin. Further, a suitable porous
material can be used in place of the porous disk. Alternatively, the top
of the column may simply be capped prior to suspension of the resin. The
total amount of resin in the column should permit a sample fluid to flow
through the column at a preferred rate of about 1-2 drops per sec.

[0109]Columns in accord with the present invention are used, for example,
as a clean-up step in analysis of liquid and solid dairy products, in
combination with detection utilizing a fluorometer. Typically, a sample
of milk is centrifuged to separate fat and a portion of the de-fatted
milk is passed over the immunoaffinity column. The top frit is optionally
removed and the resin washed several times with a methanol water
solution. At least one of the washes involves suspension of the resin
(e.g., by capping and inverting the column one or more times and
preferably several times manually or on a platform, by vortexing, by
shaking, by agitating, or by a similar method). The aflatoxins are eluted
with a methanol water solution, the eluate treated with a developing
reagent and the fluorescence read in a pre-calibrated fluorometer. Other
detection and analysis methods will occur to one of skill in the art.

[0110]It is also envisioned that the invention could be used to detect low
levels of other toxins. For example, approximately 200-250 μl of a
resin having an antibody specific for aflatoxin is layered on the disk.
For example, approximately 100-250 μl of a resin having an antibody
specific for ochratoxin is layered on the disk. For example,
approximately 250-350 μl of a resin having an antibody specific for
fumonisin is layered on the disk. For example, approximately 100-350
μl of a resin having an antibody specific for zearalenone is layered
on the disk. For example, approximately 500-550 μl of a resin having
an antibody specific for deoxynivalenol ("DON") is layered on the disk.
For example, approximately 250-350 μl of a resin having an antibody
specific for T-2 and/or HT-2 is layered on the disk. One of skill in the
art would be able to determine amounts and types of antibodies without
undue experimentation.

[0111]The column of the present invention is capable of analyzing a sample
to detect aflatoxins G1, G2, B1, B2 and M1, DON,
fumonisins B1, B2 and B3, ochratoxin A, T-2 and HT-2
toxin, and zearalenone. Such resin typically will contain about 5 mg
antibody per ml of resin. However, any suitable loading of antibody on
the resin can be used in accord with quantities and methods well known to
those skilled in the art

[0112]The total amount of resin in the column should permit a sample fluid
to flow through the column at a preferred rate of about 1-2 drops per
sec. The distance between the frits can be adjusted accordingly to allow
thorough washing of the resin and suspension as described above.

[0113]For solid foods, preferably toxins are extracted from the food using
a water-based or water compatible solvent such as, for example,
water:methanol, water:acetonitrile, ethanol, water:ethanol, salt
solutions, buffer solutions, and the like, etc. Such solvents are well
known to those skilled in the art. Typically, in such solvents the
organic component is greater. Extracts can be diluted with water prior to
chromatography.

[0114]After loading the sample on the column, the column typically is
washed to remove any extraneous materials that may be held up on the
column so that only bound materials, i.e., the toxins, remain. The column
generally can be washed with the water compatible solvent but typically
having a greater water presence. Wash solutions include, but are not
limited to, water:methanol, water:acetonitrile, ethanol, water:ethanol,
salt solutions, buffer solutions (e.g., phosphate buffered saline);
deionized water, and the like.

[0115]The volume of the resin in the column is less than the total packed
volume of the column between the lower support and the upper support. The
resin is loose within the column. During the washing step, the resin is
suspended in the wash solution, for example, by capping and inverting the
column one or more times and preferably several times manually or on a
platform, by vortexing, by shaking, by agitating, or by a similar method,
to increase the surface area of the resin which is exposed to the wash
solution. Ideally, this suspension will minimize or prevent the formation
of air pockets in the resin and/or will wash unbound, non-specific
material (which might contribute to high background readings) from the
beads prior to elution.

[0116]The column is eluted with solvents as is well known to those skilled
in the art. The eluants are analyzed for the particular analytes using
techniques including, but not limited to, photochemical analysis, post
column derivatizer, ultraviolet and fluorescent detectors.

[0117]The columns in accord with the present invention can be used as a
clean-up step in analysis of extracts from solid materials or of
colloidal or liquid products such as milk and other beverages for
aflatoxins, fumonisins, ochratoxin A, deoxynivalenol and zearalenone. The
detection of the toxin can be illustrated, typically, by spiking a sample
of a solid, extract, colloidal or liquid substance with toxins. The
sample is loaded onto the column at a speed of about 1-2 drops/second
Wash solutions include, but are not limited to, water:methanol,
water:acetonitrile, ethanol, water:ethanol, salt solutions, buffer
solutions (e.g., phosphate buffered saline), deionized water, and the
like. The toxins are eluted from the column with methanol. Detection
systems include, but are not limited to, photochemical, post-column
derivatizer, ultra-violet and fluorescence detectors. Aflatoxins are
detected by fluorescence after post-column photochemical derivitization
(post-column iodine may also be used). Fumonisin is derivatized with
o-phthaldialdehyde and detected by fluorescence. DON is detected by UV
absorbance. Zearalenone is detected by fluorescence. Ochratoxin is
detected by fluorescence. Methods for detecting the toxins are well known
to those skilled in the art.

[0118]It is desirable to obtain at least a 60%, preferably at least a 70%,
more preferably 80% recovery from the column for each toxin in the
sample. It also is desirable to have the running of the column and
detection completed within 60 minutes, more preferably within 45 minutes,
still more preferably within 30 minutes, and even still more preferably
within 25 minutes. It is also desirable to detect toxins at or below
about 500 ppt, preferably at or below about 100 ppt, more preferably at
or below about 50 ppt, even more preferably at or below about 25 ppt, and
still more preferably at or below about 12.5 ppt.

[0119]Aflatoxins include, but are not limited to, aflatoxins G1,
G2, B1, B2, and M1. Preferably, the invention
provides columns, kits, and methods for detecting levels of AFM1 at
or below about 50 ppt, even more preferably at or below about 25 ppt, and
still more preferably at or below about 12.5 ppt.

[0120]The invention is useful for detecting and measuring low levels of
other toxins and also for impurities in a wide range of substances and
not merely in milk, milk products, wine, coffee, tea, and other foods and
beverages. Rather, it is envisaged that the invention will have a broad
applicability with respect to affinity column purification.

DEFINITIONS

[0121]As used in the specification and claims, the singular form "a", "an"
and "the" include plural references unless the context clearly dictates
otherwise. For example, the term "a molecule" also includes a plurality
of molecules.

[0122]As used herein, an "analyte" is the element of the sample to be
detected or isolated. An analyte includes, but is not limited to, a
toxin, a toxoid, a toxic substance, a poisonous substance, a poison, or a
specific impurity of interest. An analyte may be of biological or
non-biological origin. In some embodiments, the analyte specifically
binds a binding reagent. In some embodiments, the presence or absence of
the analyte may be used to detect, for example, contamination of a
sample. Alternatively, the presence or absence of the analyte may be used
to determine the physiological condition of an organism from which the
sample was obtained. A wide range of other uses will occur to one of
skill in the art.

[0123]As used herein, a "toxin" includes a toxoid, a toxic substance, a
poisonous substance, or a poison of biological or non-biological origin.
In some embodiments, a toxin causes damage or disease to a cell or an
organism.

[0124]As used herein, "specificity" refers to the ability of an antibody
to discriminate between antigenic determinants. It also refers to the
precise determinants recognized by a particular receptor or antibody. It
also refers to the ability of a receptor to discriminate between
substrates, such as drugs. With respect to nucleic acids, it refers to
identity or complementarity as a function of competition or
recognition/binding, respectively. "Specificity" of recognition or
binding may be affected by the conditions under which the recognition or
binding takes place (e.g., pH, temperature, salt concentration, and other
factors known in the art).

[0125]As used herein, a "ligand" is a molecule or molecular complex that
can be bound by another molecule or molecular complex. The ligand may be,
but is not limited to, a molecule or molecular complex bound by a
receptor, or it may be a complementary fragment of nucleic acid.

[0126]As used herein, an "antibody" (Ab) is protein that binds
specifically to a particular substance, known as an "antigen" (Ag)
(described infra). An "antibody" is any immunoglobulin, including
antibodies and fragments thereof, that binds a specific epitope. The term
encompasses polyclonal, monoclonal, and chimeric antibodies (e.g.,
multispecific antibodies).

[0127]As used herein, an "antigen" (Ag) is any substance that reacts
specifically with antibodies or T lymphocytes (T cells). An
"antigen-binding site" is the part of an immunoglobulin molecule that
specifically binds an antigen.

[0128]As used herein, "biological sample" includes samples of tissues,
cells, blood, fluid, milk, or other materials obtained from a biological
organism. It also includes a biological organism, cell, virus, or other
replicative entity.

[0129]As used herein, "food" includes any substance or product intended
for human or animal consumption, regardless of nutritional value. "Food"
includes beverages, including milk and water. It also includes
ingredients or substances used in the preparation of food (e.g., oil,
fat, spices, flavorings, etc.). "Food" may be processed, partially
processed, or unprocessed. It can include artificial and/or natural
ingredients.

[0132]When not otherwise stated, "substantially" means "being largely, but
not wholly, that which is specified."

EXAMPLES

Example 1

[0133]A mobile bead column is prepared in accordance with the present
invention using resin material having a particle size in the range of
about 45 to about 165 μm with the desired antibodies attached to the
resin. A cap covers the outflow opening at the lower end of the column,
and the column is filled with an appropriate buffer known in the relevant
art. The resin is level within the column.

[0134]The cap covering the outflow opening is removed, and a liquid or
colloidal sample is applied to the column. Sample application may be
performed using a pipet, a micropipet, a medicine dropper, a funnel, a
syringe, a second column, or other means known in the art. The rate of
outflow and inflow are controlled such that the contents of the column do
not exit from the inflow opening at the upper end of the column. The flow
rate may be controlled by stopcocks at one or both ends of the column.
The flow continues until substantially all of the liquid or colloid is
passed through the column. Pressure may also be applied to the column to
control or increase the flow and speed the overall process. It may be
desirable to apply pressure when the level of the liquid or colloid
reaches the upper frit.

[0135]Preferably, the upper frit is removed and discarded. The column is
washed with a few to several volumes of an appropriate wash solution
known in the relevant art. The wash solution is allowed to flow into the
column until the column is between about one-third to about two-thirds
full. The flow is stopped, and the column is capped at both ends. The
capped column is inverted one or more times, or is shaken, such that the
resin is thoroughly suspended while being washed by the wash buffer.
After the upright position of the column is resumed, the column is
uncapped at both ends and the remaining wash solution is removed.
Additional washing cycles, with or without shaking or inversion, may be
performed. During washing, the flow rate may be faster than during
application of the sample or elution. Pressure may also be applied to the
column to control or increase the flow and speed the overall process.

[0136]After the last wash cycle, a desired amount of an elution solution
or elution buffer is applied to the column. Appropriate elution
solutions, buffers, and their desired amounts are known in the relevant
art. Typically, the volume of elution solution is minimized in order to
concentrate the eluent, which is collected and analyzed or subjected to
other procedures.

Example 2

[0137]A mobile bead column was prepared in accordance with the present
invention using Sepahrose 4B from Amersham Biosciences (Piscataway, N.J.)
with anti-aflatoxin monoclonal antibody attached to the resin to provide
about 5 mg antibody per one ml of resin. In a 3 ml column having a
diameter of 8.93 mm, a supporting porous disk (frit) was positioned at
the bottom of the column barrel in a manner to permit flow out of the
column. 250 μl of the resin was layered on the disk. Finally, another
porous disk was positioned one inch above the bottom of the column barrel
to contain the resin.

[0138]A cap was placed to cover the outflow opening at the lower end of
the column, and the column is filled with phosphate buffered saline
("PBS"; 137 mM NaCl; 2.7 mM KCl; 5.4 mM Na2HPO4; 1.8 mM
KH2PO4; pH 7.4). The side of the column was tapped to level the
resin layer so that the top of the resin layer was perpendicular to the
column.

[0139]The plunger of a 60 ml syringe was removed and discarded, and the
narrower outflow opening of the syringe barrel was fitted with a
stopcock. The syringe barrel was vertically fixed so that the wider
opening was at the top and the narrower opening with the stopcock was at
the bottom. The opposite end of the stopcock was attached with a column
coupling inserted into the upper opening of the column. With the stopcock
in a closed position, a 40 ml sample of control milk (aflatoxin
M1-free by HPLC analysis) was loaded into the wider opening at the
top of the syringe barrel. The cap covering the lower opening of the
column was removed, and the stopcock was opened so that the sample flowed
by drops into the column. The flow rate was adjusted so that the rate of
flow from the column was 1-2 drops per second.

[0140]As the upper level of the remaining sample reached the upper frit,
the rate of flow from the column slowed. The wider opening at the top of
the syringe barrel was plugged and air pressure was applied through the
plug. The process continued until the remaining milk sample had run
through the column. The upper frit was removed and discarded.

[0141]The plunger of a 10 ml syringe was removed and discarded, and the
narrower outflow opening of the syringe barrel was fitted with a stopcock
similar to the previous arrangement. 10 ml of a 10% v/v wash solution of
methanol in water was loaded into the wider opening at the top of the
syringe barrel. The rate of flow from the column was approximately 2-3
drops per second.

[0142]When the upper level of the remaining wash solution was halfway down
the column, the column was capped at the lower opening, removed, and
capped at the upper opening. The column was thoroughly inverted until all
the resin was removed from the lower frit (about ten inversions). The
column was replaced upright and the caps were removed to allow the
remaining wash solution to drain. The column was then washed twice with
10 ml aliquots of 10% methanol in water in the previous manner, but
without the inversions. The wash solution was allowed to drain
completely. (Optionally, a pump is used to speed drainage.)

[0143]An elution solution of 80% v/v methanol in water was prepared, and 1
ml was used for elution at a rate of 1-2 drops per second into a test
tube until the solution had drained completely. A 1 ml aliquot of diluted
AlfaTest® Developer (Vicam LLP; prepared from 5 ml [catalog no. 32010
with 0.03% bromine] diluted with 45 ml distilled water in accordance with
the manufacturer's instructions) was added to the eluent and then
vortexed before being placed into a precalibrated Vicam Series 4
Fluorometer and the level of AFM1 was analyzed after 60 seconds.

Additional Examples

[0144]The following examples illustrate detection of aflatoxin M1
from raw milk samples using a column containing 250 μl of a first
resin. Spiked samples were used to determine limit of detection,
linearity, precision and reproduceability.

Materials and Methods

Reagents and Chemicals

[0145]The following reagents were obtained from Vicam, LLP (Watertown,
Mass.):

[0147]AflaTest® Developer Solution: 5.0 ml AflaTest® Developer
concentrate solution (Vicam #32010) was measured and placed in the 2 oz.
amber glass bottle of a 50 ml bottle dispenser for Developer. (Vicam part
# 20600) 45.0 ml purified water was added and mixed well. The bottle
dispenser top was secured tightly. The dilute Developer solution was
capped tightly when not in use. Dilute Developer was not used more than 8
hours after preparation.

Apparatus and Equipment

[0148]The following equipment was obtained from Vicam, LLP (Watertown,
Mass.):

[0158]3.0 Sample Preparation: [0159]3.1 Approximately 50 ml milk was
centrifuged at 2000×g for 10 minutes. [0160]3.2 The skim portion
(bottom layer) of the milk was carefully removed for analysis without
disturbing the top fat layer. 40 ml of skim milk was used for each
affinity column.

[0161]4.0 Column Preparation: [0162]The Afla M1FL+ column is a
mobile resin column. Prior to use, the level of resin inside the column
was horizontal. If the resin was not already horizontal, the lower side
of the column was gently tapped several times with a finger to level the
resin.

[0163]5.0 Column Chromatography: [0164]5.1 40 ml skim milk was passed
completely through Afla M1FL+ affinity column at a rate of
about 1-2 drops/seconds until air comes through column. [0165]5.2 The
Afla M1FL+ column was removed from the loading syringe barrel.
The top frit was removed from the Afla M1FL+ column using the
Frit-picker. [0166]5.3 The Afla M1FL+ column headspace was
filled with methanol:water (10:90) solution. The Afla M1FL+
column was placed on a clean glass syringe barrel. [0167]5.4 The glass
syringe barrel was filled with 10 ml methanol:water (10:90) solution.
This solution was passed through the column at a rate of about 2-3
drops/second until the level of solution was approximately 1 inch above
the top of the resin bed.

[0168]5.5 The column was removed from the syringe barrel and capped with
the cap that came with the column. The column was inverted 10 times until
the resin beads are completely washed from the column bottom frit.
[0169]5.6 The top cap was removed from the column. The column headspace
was filled with methanol:water (10:90) and the column was placed back
onto the syringe barrel. [0170]5.7 10 ml of methanol:water (10:90) was
passed through the column at a rate of about 2-3 drops/seconds. [0171]5.8
Step 5.7 was repeated once more until air came through the column.
[0172]5.9 The affinity column was eluted by passing 1 ml methanol:water
(80:20) through column at a rate of about 1 drop/second until air came
through the column. [0173]5.10 1.0 ml of diluted AflaTest® Developer
was added to the eluate in the cuvette and mixed well. The cuvette was
placed in a calibrated fluorometer. The aflatoxin concentration was read
after 60 seconds.

[0174]6.0 Limit of Detection: At or Below 12.5 ppt

[0175]7.0 Assay Range: 0-200 ppt

Examples 3-6

Limit of Detection

[0176]For this study, the limit of detection was defined as the smallest
amount of aflatoxin M1 which is reproducibly and accurately
detected. Raw milk samples were collected from two different dairy famms,
and were determined to be aflatoxin M1-free by HPLC analysis. The
aflatoxin M1-free milk samples were spiked with aflatoxin M1
(Supelco, PA, USA) at 0, 12.5, 25, and 50 ppt. Two independent
experiments were done on two different days. The results presented in
Table 1 show the values obtained without correction for column recovery

[0180]Precision was determined using raw milk samples spiked with
aflatoxin M1 at 25 and 50 ppt. The results presented below in Table
3 (25 ppt) and Table 4 (50 ppt>were obtained from three independent
experiments, using four different raw milk samples, performed on three
different days.

[0183]Conclusion: The results above show good reproducibility at both 25
and 50 ppt levels.

[0184]Throughout this application, various publications including United
States patents, are referenced by author and year and patents by number.
The disclosures of these publications and patents in their entireties are
hereby incorporated by reference into this application in order to
describe more fully the state of the art to which this invention
pertains.

[0185]The invention has been described in an illustrative manner, and it
is to be understood that the terminology which has been used is intended
to be in the nature of words or description, rather than of limitation.

[0186]Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is, therefore,
to be understood that within the scope of the described invention, the
invention may be practiced otherwise than as specifically described.